本科毕业设计（论文）
英文文献翻译
学院名称：电气信息工程学院
专业：电子信息工程
班级：14电子1B
学号：201496111
姓名：李鹏
指导教师姓名：
指导教师职称：
二〇一六年六月
Part1：cell engineering
In 2007, Li et al. [94] proposed their microfluidic device for single cell analysis. The chip was fabricated by standard 1-photomask with low cost method. The device consists four reservoirs, four channels and one open region which contain a cell retention chamber. Reservoir one was used for cell introduction and washing, whereas reservoir two was used for reagent delivery. Reservoir three and four were used as waste reservoirs. By using this chip, they quantified dynamic Ca2+ mobilization of a single cardiomyocyte during its spontaneous contraction. Also, they monitored successfully dynamic responses from various external stimulation such as daunorubicin (cardiotoxic chemotherapeutic drug), caffeine, and isoliquiritigenin (herbal anticancer). Their results also prove that anticancer drugs have less effect on the Ca2+ of the cardiomyocytes. This device has quantified the cellular response of single cardiomyocytes, discovery of heart diseases drug and cardiotoxicity testing.
In 2010, Mellors et al. [95] proposed an electrophoretic and electrospray ionization based microfluidic device for single cell analysis. The device was fabricated on corning borosilicate glass substrate by using standard photolithography and wet chemical etching technique. Figure 12 shows the schematic of the microfluidic device, where A was a cell loading reservoir and B was buffer loading, which intersects with the separation channel
This intersection zone was a cell lysis zone. CS was an electro-osmotic pump which was connected with an electrophoretic separation channel and electrospray orifice. Cells can flow throughhydrodynamically or electrically to the intersection zone, where cells were electrically lysed. Then, cells can migrate to electrospray orifice through the separation channel where cells electrospray ionization occurred. This device successfully lysed human erythrocytes with real-time electrophoretic separation. The heme group, α and β subunits of hemoglobin were detected from erythrocytes when cells were continuously flowed through the device. This device can analyze 12 c/m.
Localized single cell membrane electroporation can provide better cell transfection
with micro/nanofluidic devices compared to single cell electroporation (SCEP) or bulk electroporation (BEP). Because of micro/nanoscale electrode dimension and distance between two electrodes were very small, as a result, electric field can intense in a very small region of the cell membrane compared to single cell dimension. Thus, the local area of the single cell can be affected by a strong electric field, whereas other areas will be unaffected. Due to the effects of small areas of the whole single cell, high cell viability and high transfection rate can be achieved compared to single cell electroporation. However, Boukany et al. show localized single cell electroporation by using nano-channel based ion transportation using electrophoresis method with two large electrodes [30]. By fabricating micro/nano electrodes with a micro/nano scale electrode gap, this device can provide some promising parameters such as low voltage and power requirement, lower toxic effect due to negligible ion generation, small sample volume and negligible heat generation. These parameters are essential to achieve high transfection rate and high cell viability. Thus, microfluidic based LSCMEP process can provide a better understanding to analyze intracellular cytosolic compounds compared to SCEP or the bulk electroporation process. Nawarathna et al., demonstrated the AFM based LSCMEP process. Figure 13 shows localized electroporation of a single cell using atomic force microscopy (AFM) technique [29]. For this experiment, they modified AFM tip to act as a nano-electrode to make an intense high electric field near the localized area of the single cell membrane. A boron doped silicon AFM tips (σ = 0.001 Ω cm, k = 1.5 N/m) was used for LSCMEP process. Before electroporation, the tip was grown with 20 nm SiO2 layer and finally this oxidized tip was sectioned until bare silicon was exposed by focused ion beam (FIB) technique. As a result, a smaller area of bare silicon can cause an intense high electric field on a single cell membrane. They have reduced this bare silicon area down to 0.5 μm in diameter, which was concentrated with an intense electric field on 10 μm diameter of rat fibroblast cell. Figure 13a–h shows the results of LSCMEP technique using AFM tip for electroporation process and Figure 13i demonstrated the AFM tip, which was positioned on top of the single cell for localized single cell membrane electroporation (LSCMEP) process. To make an intense high electric field, 1Vpp with
0.5 Hz pulse was used to transfect rat fibroblast cells. The transfection of single cell was completed within 10 s. This device can perform highly localized electroporation of a sigle cell with concentric electric field on local area of single cell membrane. The experiment can be performed in a friendly environment such as cell culture dishes, etc.
In recent years, Boukany et al. [30] showed nanochannel based localized single cell electroporation with a precise amount of biomolecules delivery. In this device, they positioned a single cell in one microchannel by optical tweezers and transfection agent was loaded to another microchannel. These two microchannels were connected by one nanochannel. To apply a very high electric field in between two microchannels, a transfection agent was delivered through the nanochannel using an electrophoretically driven process and finally drugs were delivered inside a single cell through a very small area of the cell membrane. In 2012, Chen et al. demonstrated another localized single cell membrane electroporation usimg ITO microelectrode based transparent chip [27]. Figure 14 shows microfluidic localized single cell membrane electroporation device. They deposited ITO films on a covered glass substrate and patterend it by standard lithographic process to form as ITO lines. After that, a thin SiO2 layer was deposited as a passivation layer by plasma enhanced chemical vapor deposition (PECVD) technique. The final ITO lines were cut by the focused ion beam (FIB) technique. The gap between two electrodes were 1 μm and width of each electrode was 2 μm. When single cell was strongly attached in be tween two electrodes gap, the electric field was intensed in only a 1 μm gap area on single cell membrane. As a result, they demonstrated localized single cell membrane electroporation with microfluidic device. Figure 14a shows localized electroporation process between two micro-electrodes and Figure 14b shows multiple number of electrodes for LSCMEP process. Figure 14 c and d shows the optical microscope image of patterened ITO microelectrodes and scanning electron microscope (SEM) image of ITO microelectrodse with micro-channel. According to their results, they achived 0.93 μm electroporation region with 60% cell viability for 8Vpp 20 ms pulse application. To reduce the gap between two electrodes, a high transfection rate can be
achived by this technique. This device not only control the recovery of cell membranes (reversible electroporation) without cell damage but also it provides clear optical view by using an inverted microscope (ITO based transparent chip). Recently, another LSCMEP based device was proposed by Jokilaakso et al. [77] for single cell lysis. They reported a silicon nanowire and nanoribbon based biological field effect transistor for single cell positioning and lysis mechanism. Figure 15a shows the cross sectional view and electric connection with PDMS above the device and Figure 15b shows an array of the transistors with both nanowires and nanoribons. To position the single cell on this device, they used programmable magnetic field for magnetic manipulation of 7.9 μm COOH modified COMPEL magnetic microsphere. After positioning the single cell (HT-29) on top of the transistor, cells were adhered for 30 min prior to electroporation experiment. The applied electric field was 600–900 mVpp (peak to peak) at 10 MHz for 2 ms pulse. This electric field was connected with a shorted source and drain in one terminal and another terminal connected on the gate of the device. The electric field intensity was fringing in nature, which affected the cell membrane integrity leading to cell lysis. This device can perform single cell lysis which is potentially applicable to medical diagnostics and biological cell studies.
In summary, this article describes the details about bulk electroporation (BEP), single cell electroporation (SCEP), and localized single cell membrane electroporation (LSCMEP) by using micro/nanofluidic devices with their advantages and disadvantages. All of these processes can deliver drugs, DNA, RNA, oligonucleotides, proteins, etc. However, to analyze cell to cell behavior with their organelles and intracellular biochemical effect, single cell analysis must be executed. Micro/nanofluidic devices are the potential candidates to analyze single cells, because of their dimension reduction to the dimension of single cell level. These devices provide easy performance such as cell handling, lower power consumption, low toxicity, small sample volume, lower contamination rate, high cell viability, and high transfection rate when compared to conventional electroporation. To reduce the electrode area and gap between two electrodes by using micro/nanofluidic devices,
selective and localized drug delivery is possible. This new approach is called localized single cell membrane electroporation (LSCMEP). However, until now this technique is in the development stage. In the future, the LSCMEP process can provide selective and specific single cell manipulation from millions of populations of cells together. Micro/nanofluidic devices can approach this level in the near future, which will be potentially beneficial for medical diagnostics, proteomics analysis and biological studies.
Part2：Nerve and muscle cells
2.1 INTRODUCTION
In this chapter we consider the structure of nerve and muscle tissue and in particular their membranes(动植物体内的隔膜、薄膜), which are excitable. A qualitative description of the activation process follows. Many new terms and concepts are mentioned only briefly in this chapter but in more detail in the next two chapters, where the same material is dealt with from a quantitative rather than a qualitative point of view.
The first documented reference to the nervous system is found in ancient Egyptian records. The Edwin Smith Surgical Papyrus, a copy (dated 1700 B.C.) of a manuscript composed about 3500 B.C., contains the first use of the word "brain", along with a description of the coverings of the brain which was likened to the film （薄层、薄膜）and corrugations（起皱、皱纹）that are seen on the surface of molten copper（熔融铜）as it cooled (Elsberg, 1931; Kandel and Schwartz, 1985).
The basic unit of living tissue is the cell. Cells are specialized in their anatomy and physiology to perform different tasks. All cells exhibit a voltage difference across the cell membrane. Nerve cells and muscle cells are excitable. Their cell membrane can produce electrochemical impulses and conduct them along the membrane. In muscle cells, this electric phenomenon is also associated with the contraction of the cell. In other cells, such as gland cells and ciliated cells, it is believed that the membrane voltage is important to the execution of cell function.
The basic unit of living tissue is the cell. Cells are specialized in their anatomy and physiology to perform different tasks. All cells exhibit a voltage difference across the cell membrane. Nerve cells and muscle cells are excitable. Their cell membrane can produce electrochemical impulses and conduct them along the membrane. In muscle cells, this electric phenomenon is also associated with the contraction of the cell. In other cells, such as gland cells and ciliated cells, it is believed that the membrane voltage is important to the execution of cell function.
The origin of the membrane voltage is the same in nerve cells as in muscle cells. In both cell types, the membrane generates an impulse as a consequence of excitation. This impulse propagates in both cell types in the same manner. What follows is a short introduction to the anatomy and physiology of nerve cells. The reader can find more detailed information about these questions in other sources such as Berne and Levy (1988), Ganong (1991), Guyton (1992), Patton et al. (1989) and Ruch and Patton (1982).
2.2 NERVE CELL
2.2.1 The Main Parts of the Nerve Cell
The nerve cell may be divided on the basis of its structure and function into three main parts:
(1) the cell body, also called the soma;
(2) numerous short processes of the soma, called the dendrites; and,
(3) the single long nerve fiber, the axon.
Fig. 2.1. The major components of a neuron.
The body of a nerve cell (see also (Schad and Ford, 1973)) is similar to that of all other cells. The cell body generally includes the nucleus, mitochondria, endoplasmic reticulum, ribosomes, and other organelles. Since these are not unique to the nerve cell, they are not discussed further here. Nerve cells are about 70 - 80% water; the dry
material is about 80% protein and 20% lipid. The cell volume varies between 600 and 70,000.
The short processes of the cell body, the dendrites, receive impulses from other cells and transfer them to the cell body (afferent signals). The effect of these impulses may be excitatory or inhibitory. A cortical neuron (shown in Figure 2.2) may receive impulses from tens or even hundreds of thousands of neurons (Nunez, 1981).
The long nerve fiber, the axon, transfers the signal from the cell body to another nerve or to a muscle cell. Mammalian axons are usually about 1 - 20 in diameter. Some axons in larger animals may be several meters in length. The axon may be covered with an insulating layer called the myelin sheath, which is formed by Schwann cells (named for the German physiologist Theodor Schwann, 1810-1882, who first observed the myelin sheath in 1838). The myelin sheath is not continuous but divided into sections, separated at regular intervals by the nodes of Ranvier (named for the French anatomist Louis Antoine Ranvier, 1834-1922, who observed them in 1878).
2.2.2 The Cell Membrane
The cell is enclosed by a cell membrane whose thickness is about 7.5 - 10.0 nm. Its structure and composition resemble a soap-bubble film (Thompson, 1985), since one of its major constituents, fatty acids, has that appearance. The fatty acids that constitute most of the cell membrane are called phosphoglycerides. A phosphoglyceride consists of phosphoric acid and fatty acids called glycerides (see Figure 2.3). The head of this molecule, the phosphoglyceride, is hydrophilic (attracted to water). The fatty acids have tails consisting of hydrocarbon chains which are hydrophobic (repelled by water).
Fig. 2.3. A sketch illustrating how the phosphoglyceride (or phospholipid) molecules behave in water. See text for discussion.
翻译一：细胞工程
在2007年。李某提出了单细胞分析的微流控装置。芯片是依据标准1-photomask制造的，并且成本低。装置包括四个水库，四个通道和一个能使细胞滞留的开放区域。一个水库用于细胞的引入，然而两个水库用于试剂的传送。水库三和四则用于废液的储存。通过使用芯片，他们确定单个心肌细胞的动态Ca2+的数量，及在其中是自发的收缩。并且，他们成功地监测的动态响应各种外部刺激如柔红霉素（心脏毒性的化疗药物），咖啡因，和异甘草素（草本抗癌）。他们的研究结果也表明抗癌药物对心肌细胞内的Ca 2+影响较小。这个装置具有确定单个心肌细胞的细胞反应的数量,发现心脏疾病的药物和毒性测试。
在2010年，Mellors等提出了基于单细胞分析微流控装置为基础的一个电泳和电喷雾电离。芯片是依据在康宁硼硅玻璃板上使用标准光刻和湿化学法刻蚀技术。图12显示了微流控装置的示意图,A是一个细胞加载水库，而B是缓冲加载,并且是与分离相交的通道。
这个交叉区是一个细胞裂解区。CS是电渗泵连接一个电泳分离通道和电喷雾孔。细胞可以在水动力或电的作用下游动到交叉区域,发生细胞被电裂解。然后,细胞可以迁移到电喷雾孔通过分离通道细胞电喷雾电离发生的地方。这个装置成功地裂解人类红细胞，实时的电泳分离。当细胞连续的流过装置时，能够检测到红细胞中血红蛋白的α和β亚基（血红素组红细胞）。这个装置可以分析12 c / m。
局部单细胞膜电穿孔可以比单个细胞电穿孔(SCEP)或散装电穿孔(EP)提供更好的微纳
流控装置的细胞转染。由于微/纳米电极尺寸和两个电极之间的距离非常小,因此,相比于单细胞尺寸，电场可以在一个非常小的的细胞膜区域更加强烈。因此,局部的单细胞可以影响一个强大的电场,而其他地区将不会受到影响。由于对整个细胞小面积的影响，与单细胞电穿孔相比,细胞具有更高的细胞活力和转染率。然而,Boukany等表明局部单细胞电穿孔通过使用纳米通道基于离子运输用电泳法两大电极。通过与微/纳米尺度制造微/纳米电极的电极间隙,这个装置可以提供一些有前途的低电压和功率要求等参数
要求,由于离子生成可以忽略不计，故可以降低毒性作用；由于样本体积小，故可以忽略不计的发热。这些参数与实现细胞高得转染率和高的细胞活力有重要关联。因此,基于微流体LSCMEP过程可以提供一个更好的理解分析细胞内胞浆化合物，与SCEP或散装的电穿孔过程相比。Nawarathna等基于LSCMEP过程证明了原子力显微镜。图13显示单细胞的局部电穿孔使用原子力显微镜(AFM)技术。在这个实验中,他们修改了AFM针尖作为纳米电极，进行强烈的高电场附近的单细胞膜局部区域。硼掺杂的硅针尖(σ= 0.001Ω厘米,k= 1.5 N / m)被用于LSCMEP过程。在电穿孔前, 尖端20纳米SiO2层和最后尖端生长氧化硅切片到赤裸的采用聚焦离子束（FIB）技术暴露。结果,小面积裸露的硅能引起一场高强度电场在单细胞膜上。他们减少了裸露的硅面积降至直径为0.5μm，与高强度电场反应，集中在直径为10μm鼠成纤维细胞上。图13a–H显示使用AFM针尖电穿孔过程的LSCMEP的结果和图13i显示原子力显微镜的尖端，这是位于顶部的单细胞局部单细胞膜电穿孔（LSCMEP）过程。为了做一个高强度的电场,1 vpp、 0.5赫兹脉冲被用来转染鼠成纤维细胞。单细胞的转染在10s内完成。这个装置可以高度地执行单细胞局部的电穿孔，随着同心电场在单细胞膜区域执行。这个实验可以在友好的环境中执行,如细胞培养等。
近年来,Boukany等表明基于局部单细胞电穿孔纳米通道的与精确数量的生物分子传送。在这个装置中,他们将单细胞定位在微通道的光学镊子及将转染剂加载到另一个微通
道。这两个微通道由一个纳米通道连接。在两个微通道中加入一个高强度的电场，转化剂的传送通过纳米通道使用用电泳驱动过程通道，最后的药物传送到单细胞通过细胞膜的一个很小的区域。2012年，chen等展示了另一个局部单细胞膜电穿孔基于ITO微电极的透明芯片。图14显示微流控局部单细胞膜电穿孔装置。他们使ITO薄膜沉积在玻璃上,patterend通过标准光刻形成ITO线。在那之后,一层薄薄的SiO
层是沉积钝化层
2
的等离子体增强化学气相沉积(PECVD方法)技术。最后ITO线通过聚焦离子束(FIB)技术删减。两个电极之间的距离为1μm，每个电极的宽度是2μm。当单细胞强烈的连接在两个电极之间,电场增强只有1μ米落差地区单细胞膜。结果,他们证明了局部单细胞膜电穿孔与微流控装置。图15a展示两个微型电机和之间的局部电穿孔过程，图14b显示电极的多个数之间的lscmep过程。图14 C和D显示patterened ITO电极和扫描电子显微镜（SEM）图像的光学显微镜的ITO microelectrodse微通道的图像。根据他们的结果，他们取得了0.93μM电穿孔区与8vpp 20毫秒脉冲应用60%细胞活力。减少在两个电极之间的间隙，高转染率可以通过这种技术实现。该装置不仅控制了细胞膜的回收（可逆电穿孔）的无细胞的损伤也提供了明确的光学角度采用倒置显微镜（ITO透明片）最近,另一个LSCMEP基器件是由 Jokilaakso等提出的针对单细胞裂解。他们报道的硅纳米线和纳米带为基础的生物场效应晶体管的单细胞定位和裂解机理。图15a显示了横断面视图和PDMS高于设备和电气连接，图15b显示的纳米线和nanoribons晶体管阵列。这个装置上单细胞的位置可用可编程的磁场为7.9μm磁操纵羧基改性强迫磁性微球。定位后的单细胞(HT-29)上的晶体管,细胞电穿孔试验之前粘附了30分钟。应用的电场为(峰间)在10 MHz ，2毫秒脉冲。这个电场与短路的源极和流失在一个终
600 - 900 mV
pp
端和另一个终端连接装置的门。在本质上，电场强度的边缘影响了细胞膜完整性导致细胞裂解。这个装置可以执行单细胞裂解作用可能是医学诊断和生物细胞研究潜在的应用。
总之,本文描述了大部分电穿孔(cep)的细节,单细胞电穿孔(SCEP)和局部单细胞膜电穿孔(LSCMEP)通过使用微/纳米流体电设备的优点和缺点。所有这些过程可以提供药
物,DNA,RNA,寡核苷酸,蛋白质等。然而,分析细胞之间的行为与他们的细胞器和细胞内的生化作用，单细胞分析必须执行。微/奈米流体电装置是分析单细胞的潜在候选,因为他们的降维法在单细胞水平的尺寸。这些装置提供简单的性能如细胞处理,低功耗,低毒性,小样本体积,降低污染率,高细胞生存能力和高转染率相比,传统的电穿孔。减少电极面积和两个电极之间的差距通过使用微/奈米流体电设备,选择性和局部给药是可能的。这种新方法叫做局部单细胞膜电穿孔(LSCMEP)。然而,直到现在这种技术仍在发展阶段。将来,LSCMEP过程可以提供选择性和特定的单细胞操纵从数百万的细胞中。在不久的将来，微/奈米流体电装置可以达到这个水平,将有可能有利于医学诊断、蛋白质组学分析和生物研究。
翻译二：神经和肌肉细胞
2.1 引言
在这一章中, 我们会了解神经和肌肉组织的结构,尤其是那些容易激动的隔膜或薄膜. 下面是对激化过程的一个定性的描述. 许多新的术语和概念会在这一章中简单的提到,至于更详细的内容将会在后面两章中讲述, 而且相同的内容会用定量的方法解决而不是定性的方法了.
最早涉及神经系统的文献被发现于古埃及人的记录中. 爱德华·史密斯的《Surgical Papyrus》，一份（公元前1700年）源于约公元前3500年的手稿的副本，包含了“brain”的第一次使用，有这样一描述：大脑被比作起皱的薄层，就像冷却以后的的熔融铜的表面一样。
活体组织的基本单位是细胞，细胞在解剖学和生理学方面扮演者各种各样的任务。所有的细胞隔着细胞膜有一个电位差。神经和肌肉细胞的电位是容易激发的。他们的细胞膜产生电化学脉冲，并且可以沿着细胞膜传导。在肌细胞中，这些电现象还跟细胞的收缩有关。在其他细胞，像腺细胞和纤毛细胞，据认为膜电位对于细胞功能的执行也是重要的。
膜电位的起源在神经细胞和肌细胞中是一样的。在两种细胞类型中，细胞膜都是由于刺激产生了一个脉冲。这种冲在两种细胞中是以相同的方式传播的。下面是对神经细胞解剖学和生理学的一个简短的介绍。读者也可以在其他资料上找到有关这些问题更详细的信息，像Berne and Levy (1988), Ganong (1991), Guyton (1992), Patton et al.
2.2 神经细胞
2.2.1 神经细胞的主要内容：
基于结构和功能，神经细胞可以划分为三类：
（1）细胞体，也叫体细胞
（2）许多未加工的体细胞，叫树突
（3）单一长神经纤维，叫轴突。
以下是对图2.1的描述：
神经细胞的胞体和其他细胞的胞体是相似的。细胞体大体上包括细胞核，线粒体，内质网，核糖体和其他细胞器。既然这些对于神经细胞不是独一无二的，那么这里将不做深入的探讨。神经细胞70%~80%是水；干物质中80%的蛋白质和20%的脂质。细胞的体积从600~7000不等。
图2.1 神经元的主要构成
缺少加工的细胞体，即树突，可以接受来自其他细胞的冲动并且把他们传到细胞体（传入信号）。这些冲动的效应可以是兴奋性的，也可以是抑制性的。大脑皮层神经元可以接受来自几十几百甚至上千的神经元的冲动。
长神经纤维，即轴突，把信号从一个胞体传递到另一个神经细胞或肌肉细胞。哺乳动物轴突的直径大约是1~2 。一些更大的动物可以长达几米。轴突被一层叫做髓鞘的绝缘层所包裹，它是由施沃恩细胞形成的（因德国生理学家Theodor Schwann, 1810-1882,而得名，他首次在1838年发现了髓鞘）。髓鞘不是连续的，而是被郎维结（因法国解剖学家Louis Antoine Ranvier, 1834-1922,的名，他在1878年发现了郎维结）分成一段一段的零件。
2.2.2 细胞膜
细胞表面被附了一层厚度为7.5~10.0纳米的细胞膜。它的组成结构像一个肥皂泡似的薄膜，由于它主要的一种组份，脂肪酸，使它呈现这种面貌。这些构成细胞膜的脂肪酸叫磷酸甘油酯。磷酸甘油酯由磷酸和甘油酯（脂肪酸丙酯）（如图2.3）组成。这个分子的头部是磷酸甘油酯，它是亲水的。尾部是包含烃链的
憎水性脂肪酸。
图2.3. 一幅说明磷脂分子在水中行为的图，讨论见文本。